Compounds and compositions comprising fluorophores for use in visualization and purification or manipulation

Abstract

The application discloses a compound having a moiety that is either an affinity tag-containing moiety or a protein-manipulation moiety, a self-labeling protein (SLP) ligand, and a rhodamine dye linking the affinity tag-containing moiety or a protein-manipulation moiety to the SLP ligand. Also disclosed is a complex, which includes the compound and a SLP. Also disclosed is a method that involves contacting the compound and a SLP with a cell, and visualizing fluorescence in the cell or purifying the SLP and associated biological components from the cell.

What is claimed is: 
     
         1 . A compound, according to the formula: 
       
         
           
           
               
               
           
         
         or a salt thereof, wherein:
 X is O, C(CH 3 ) 2 , or Si(CH 3 ) 2 ; 
 R 1  is an affinity tag-containing moiety or a protein-manipulation moiety; 
 each R 2  is independently selected from the group consisting of H, D, halogen, OH, O(alkyl), N(alkyl) 2 , CF 3 , CN, COOH, COO(alkyl), C(O)NH(alkyl), C(O)N(alkyl) 2 , and SO 2 CH 3 ; 
 each R 3  is independently selected from the group consisting of H and D; 
 each R 4  is independently selected from the group consisting of H, halogen, CF 3 , and CN; and 
 R 5 , R 6 , R 7 , and R 8  are each independently selected from the group consisting of H, F, CO 2 H, and a self-labeling protein (SLP) ligand, so long as one of R 5 , R 6 , R 7 , and R 8  is the SLP ligand. 
 
       
     
     
         2 . The compound of  claim 1 , wherein R 1  is 
       
         
           
           
               
               
           
         
       
     
     
         3 . The compound of  claim 2 , wherein the SLP ligand is 
       
         
           
           
               
               
           
         
       
     
     
         4 . The compound of  claim 1 , wherein the SLP ligand is 
       
         
           
           
               
               
           
         
       
     
     
         5 . The compound of  claim 2 , wherein R 2  is H or F; R 3  and R 4  are H; R 5 , R 6 , and R 8  are H; and R 7  is the SLP ligand. 
     
     
         6 . The compound of  claim 5 , wherein X is O, Si(CH 3 ) 2 , or Si(CH 3 ) 2 . 
     
     
         7 . The compound of  claim 1 , wherein X is O, Si(CH 3 ) 2 , or Si(CH 3 ) 2 . 
     
     
         8 . The compound of  claim 5 , wherein the SLP ligand is 
       
         
           
           
               
               
           
         
       
     
     
         9 . The compound of  claim 1 , of the following formula: 
       
         
           
           
               
               
           
         
         wherein R is H or F; X is O, C(CH 3 ) 2 , or Si(CH 3 ) 2 ; and L is an affinity tag-containing moiety or a protein-manipulation moiety. 
       
     
     
         10 . The compound of  claim 1 , of the following formula: 
       
         
           
           
               
               
           
         
         wherein R is H or F, and X is O, C(CH 3 ) 2 , or Si(CH 3 ) 2 . 
       
     
     
         11 . The compound of  claim 1 , of the following formula: 
       
         
           
           
               
               
           
         
         
           
           
               
               
           
         
       
     
     
         12 . The compound of  claim 1 , of the following formula: 
       
         
           
           
               
               
           
         
       
     
     
         13 . The compound of  claim 1 , of the following formula: 
       
         
           
           
               
               
           
         
       
     
     
         14 . The compound of  claim 1 , wherein R 1  is an affinity tag-containing moiety consisting of an affinity tag and a linker joining the affinity tag to the compound. 
     
     
         15 . The compound of  claim 14 , wherein the affinity tag is selected from the group consisting of biotin, desthiobiotin, trimethoprim/folate/methotrexate, a peptide epitope, or a click chemistry reagent. 
     
     
         16 . The compound of  claim 15 , wherein the linker is selected from the group consisting of a polyethylene glycol (PEG) bearing a terminal amino group, an alkane bearing a terminal amino group, a polypeptide, or a maleimide-thiol group, or an azide-alkyne click chemistry group. 
     
     
         17 . The compound of  claim 1 , wherein R 1  is a protein-manipulation moiety consisting of a protein-manipulation ligand and a linker joining the protein-manipulation ligand to the compound. 
     
     
         18 . The compound of  claim 17 , wherein the protein-manipulation ligand is an inhibitor, allosteric binder, or activator. 
     
     
         19 . The compound of  claim 18 , wherein the linker is selected from the group consisting of a polyethylene glycol (PEG) bearing a terminal amino group, an alkane bearing a terminal amino group, a polypeptide, or a maleimide-thiol group, or an azide-alkyne click chemistry group. 
     
     
         20 . A complex, comprising: a compound of  claim 1 , and further comprising a self-labeling protein (SLP). 
     
     
         21 . A method, comprising: contacting the compound of  claim 1  and a self-labeling protein (SLP) with a cell; and visualizing fluorescence in the cell or purifying the SLP and associated biological components from the cell.